The present invention generally relates to a method for using calibration beads to enhance the performance of a flow imaging system, and more specifically, to using calibration beads to facilitate the reliable collection of velocity data used by the flow imaging system.
Cells and cell groupings are three-dimensional objects containing rich spatial information. The distribution of a tremendous variety of bio-molecules can be identified within a cell using an ever-increasing number of probes. In the post-genome era, there is mounting interest in understanding the cell, not only as a static structure, but as a dynamic combination of numerous interacting feedback control systems. This understanding can lead to new drugs, better diagnostics, more effective therapies, and better health care management strategies. However, this understanding will require the ability to extract a far greater amount of information from cells than is currently possible.
The principal technologies for cellular analysis are automated microscopy and flow cytometry. The information generated by these mature technologies, although useful, is often not as detailed as desired. Automated microscopy allows two-dimensional (2D) imaging of from one to three colors of cells on slides. Typical video frame rates limit kinetic studies to time intervals of 30 ms.
Instruments known as flow cytometers currently provide vital information for clinical medicine and biomedical research by performing optical measurements on cells in liquid suspension. Whole blood, fractionated components of blood, suspensions of cells from biopsy specimens and from cell cultures, and suspensions of proteins and nucleic acid chains are some of the candidates suitable for analysis by flow cytometry. In flow cytometers specialized for routine blood sample analysis, cell type classification is performed by measuring the angular distribution of light scattered by the cells and the absorption of light by specially treated and stained cells. The approximate numbers of red blood cells, white blood cells of several types, and platelets are reported as the differential blood count. Some blood-related disorders can be detected as shifts in optical characteristics, as compared to baseline optical characteristics, such shifts being indicative of morphological and histochemical cell abnormalities. Flow cytometers have been adapted for use with fluorescent antibody probes, which attach themselves to specific protein targets, and for use with fluorescent nucleic acid probes, which bind to specific DNA and RNA base sequences by hybridization. Such probes find application in medicine for the detection and categorization of leukemia, for example, in biomedical research, and drug discovery. By employing such prior art techniques, flow cytometry can measure four to ten colors from living cells. However, such prior art flow cytometry offers little spatial resolution, and no ability to study a cell over time. There is clearly a motivation to address the limitations of existing cell analysis technologies with a novel platform for high speed, high sensitivity cell imaging.
A key issue that arises in cell analysis carried out with imaging systems is the measurement of the velocity of a cell or other object through the imaging system. In a conventional time-domain methodology, cell velocity is measured using time-of-flight (TOF). Two detectors are spaced a known distance apart and a clock measures the time it takes a cell to traverse the two detectors. The accuracy of a TOF measurement is enhanced by increasing detector spacing. However, this increases the likelihood that multiple cells will occupy the measurement region, requiring multiple timers to simultaneously track all cells in view. Initially, the region between the detectors is cleared before starting sample flow. As cells enter the measurement region, each entry signal is timed separately. The system is synchronized with the sample by noting the number of entry signals that occur before the first exit signal.
TOF velocity measurement systems are prone to desynchronization when the entry and exit signals are near threshold, noise is present, or expected waveform characteristics change due to the presence of different cell types and orientations. Desynchronization causes errors in velocity measurement that can lead to degraded signals and misdiagnosed cells until the desynchronized condition is detected and corrected. Resynchronization may require that all cells be cleared from the region between the detectors before restarting sample flow, causing the loss of sample.
Significant advancements in the art of flow cytometry are described in commonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001, and entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955, issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawings of each of these patents are hereby specifically incorporated herein by reference.
The inventions disclosed in the above noted patents perform high resolution, high-sensitivity two-dimensional (2D) and three-dimensional (3D) imaging using time-delay-integration (TDI) electronic image acquisition with cells in flow. These instruments are designed to expand the analysis of biological specimens in fluid suspensions beyond the limits of conventional flow cytometers. TDI sensors utilize solid-state photon detectors such as charge-coupled device (CCD) arrays and shift lines of photon-induced charge in synchronization with the flow of the specimen. The method allows a long exposure time to increase a signal-to-noise ratio (SNR) in the image while avoiding blurring. However, precise synchronization of the TDI detector timing with the motion of the moving targets is required. For example, if a target is to traverse 100 lines of a TDI sensor to build an image, and the blurring is expected to be less than a single line width, then the velocity of the target must be known to less than one percent of its actual value. It would thus be desirable to provide method and apparatus capable of producing highly accurate flow velocity for such moving targets.
Several methods for determining velocity for use in such flow imaging instruments are described in commonly assigned, copending application entitled MEASURING THE VELOCITY OF SMALL MOVING OBJECTS SUCH AS CELLS, Ser. No. 09/939,292, filed on Aug. 24, 2001, the specification and drawings of which are hereby specifically incorporated by reference.
Proper functioning of flow imaging systems that require the synchronization of a TDI detector with objects in flow requires consistent and reliable velocity information. This can be particularly difficult to achieve when the fluid flow contains only a small number of particles, when only a small volume of sample fluid is available, when only limited amounts of light from target cells are available, and when a distribution of target cells in a sample is uneven. It would be desirable to provide a method for determining reliable velocity data under such conditions. It would further be desirable to provide methods to facilitate diagnostic and calibration procedures for flow imaging systems.
The present invention is a method for utilizing calibration beads to enhance the performance of a flow imaging system. Related applications have described preferred flow imaging systems and preferred methods of measuring the velocity of objects in flow passing through such systems. Such imaging systems are beneficially employed to produce images of objects of interest, such as biological cells.
Non sample particles, referred to as calibration beads, can be introduced into a flow of fluid in such a flow imaging system for the purpose of establishing a velocity. Such calibration beads are preferably polymer micro spheres, but it should be understood that substantially any object capable of being suspended in a fluid, and whose dimensions are compatible with the imaging system being employed, can be utilized as a calibration bead. With respect to the dimensions, such calibration beads must be small enough to pass through the flow cell of the imaging system without obstruction, and yet large enough to be readily detectable by the imaging systems optics and sensors. An optimal size calibration bead for a first imaging system may not represent an optimal size for a second imaging system. Examples of particles that can be beneficially employed as calibration beads include cells, cell clusters, labeled and unlabelled micro spheres (polymer, copolymer, tetra polymer and silica beads).
The use of such calibration beads is particularly helpful when the fluid flow contains only a small number of particles, when only a small volume of sample fluid is available, when only limited amounts of light from target cells are available, when a distribution of target cells in a sample is uneven, and to facilitate diagnostic and calibration procedures.
When the fluid flow contains only a small number of particles it is useful to employ a relatively high concentration of calibration beads, to enable the continuous detection of flow speed velocity. The continuous velocity measurement enables continuous TDI detector/flow speed synchronization, enhancing the stability and performance of flow imaging systems. When a sample particle is imaged, the stable TDI detector/flow speed synchronization facilitates the collection of more precise sample data than can be achieved in an imaging system with poor TDI detector/flow speed synchronization. A preferred concentration of calibration beads will be selected based on parameters of the flow imaging system being employed, to ensure that sufficient calibration beads are provided so that the velocity of the fluid in the flow cell is continually monitored.
Another circumstance in which the use of calibration beads can facilitate accurate velocity measurements is when the actual volume of the sample is small. A fluid flow of calibration beads (i.e. no sample) can be employed to initialize a flow imaging system, and to establish a stable hydrodynamically focused fluid flow in the flow cell of such an imaging system. The calibration beads enable the TDI Camera/Velocity synchronization to be established. When the imaging system and fluid flow is stable, the sample containing the objects of imaging interest can be introduced into the flow cell for analysis of the sample objects.
The amount of light from an object corresponds to the precision of the velocity measurement. Lower levels of light provide less precise velocity data. Often objects of interest (i.e.) samples are so small that while they do provide sufficient light to generate an image, the velocity data obtainable is less precise than desired, thereby making accurate TDI synchronization with the fluid flow difficult. As the signal strength of the light from the objects is generally proportional the size of the object, calibration beads that are larger than the anticipated size of the objects of interests can be employed to increase velocity detection resolution.
In a related problem, samples can include several different types of objects of interest, each of which are of different sizes or properties, and each of which provide different levels of light from which images and velocity data can be obtained. Different levels of light correspond to different levels of precision in determining velocity, which in turn means that the TDI synchronization with the fluid flow can undesirably vary. Adding calibration beads to such a sample volume provides a consistent velocity signal, enabling TDI synchronization to be more reliably maintained.
When utilized in a flow imaging instrument, calibration beads can also provides a known data source that can be employed in various self-diagnostic, calibration and quality metric applications for the both the optical system of the flow imaging instrument, as well as the flow cell of the flow imaging instrument. Such data can be used to determine point spread functions associated with an imaging system, to determine a sensitivity of an imaging system, and to determine a focal point of the imaging system. Imagery collected from calibration beads can be used to determine core size and stability and TDI/flow speed synchronization.
Preferred calibration beads are polymeric beads, including but not limited to following types: polystyrene, styrene/divinylbenzene copolymer (S/DVB), polymethylmethacrylate (PMMA), polyvinyltoluene (PVT), styrene/butadiene (S/B), styrene/vinyltoluene (S/VT). Mixtures of different types of calibration beads may be used. Preferable calibration beads will have densities in the range of 0.9-2.3 grams per cubic centimeter, and diameters that range from 20 nanometers to 50 microns. Calibration beads may incorporate surface functional groups enabling the covalent coupling of ligands. Such surface functional groups preferably include: sulfate based groups (xe2x80x94SO4), aldehyde based groups (xe2x80x94CHO), aliphatic amine based groups (xe2x80x94CH2xe2x80x94NH2), amide based groups (xe2x80x94CONH2), aromatic amine based groups (xe2x80x94NH2), carboxylic acid based groups (xe2x80x94COOH), chloromethyl based groups (xe2x80x94CH2xe2x80x94Cl), hydrazide based groups (CONHxe2x80x94NH2), hydroxyl based groups (xe2x80x94OH), and sulfonate based groups (xe2x80x94SO3). Calibration beads can be beneficially incorporate a coating of protein A or streptavidin. Further, calibration beads can include dyed microspheres of different colors, fluorescent labeled microspheres, magnetic microspheres, and molecularly imprinted micro spheres.